Oxidized coenzyme Q10, which is a benzoquinone derivative widely distributed in the living world, is also called vitamin Q because of its vitamin-like function and is an ingredient acting as a nutrient in restoring the cell activity that has been weakened to its healthy condition and rejuvenating a living body. On the other hand, reduced coenzyme Q10 is derived from oxidized coenzyme Q10 by two-electron reduction. Reduced coenzyme Q10 is a white crystal whereas oxidized coenzyme Q10 is an orange crystal. Reduced coenzyme Q10 and oxidized coenzyme Q10 are known to be localized in mitochondrion, lysosome, Golgi body, microsome, peroxisome, cell membrane, etc., and involved, as constituents of the electron transport system, in ATP production and activation, antioxidant activity in a living body, and membrane stabilization. They are thus substances indispensable for maintenance of living body functions.
Reduced coenzyme Q10 is readily oxidized to oxidized coenzyme Q10 by a molecular oxygen. Complete oxygen elimination or blocking is very difficult to be achieved in a commercial scale production, preservation or handling and, furthermore, fairly long periods of time are required for individual operations, unlike a laboratory scale production. Thus, residual oxygen exerts great adverse effects such as oxidation of reduced coenzyme Q10 to oxidized coenzyme Q10. As described above, it is difficult to obtain a reduced coenzyme Q10 crystal of high quality on a commercial scale. Even if reduced coenzyme Q10 of high quality could be produced, oxidation stability of reduced coenzyme Q10 is a very important subject in processing them into foods, functional nutritive foods, specific health foods, nutrients, nutritional supplements, animal drugs, drinks, feeds, cosmetics, medicines, remedies, preventive drugs, etc., or into materials and compositions thereof, and/or in preserving them after the process. Also in the process and the preservation mentioned above, complete oxygen elimination or blocking is quite difficult. Particularly in heating during the process or preservation for a long period of time, residual or immixing oxygen exerts a great adverse effect. Thus, protection against oxidation in the above-mentioned production, keeping, handling, process and preservation is very important. Oxidized coenzyme Q10, which is a byproduct yielded by oxidation of the above reduced coenzyme Q10, decreases a yield of reduced coenzyme Q10. Furthermore, oxidized coenzyme Q10 is difficult to be separated from reduced coenzyme Q10, therefore immixes in the reduced coenzyme Q10 product as an impurity and thereby decreases purity or makes the obtained crystal yellowish. Consequently, a problem that consumers or customers feel a sense of discomfort, or the like problem occurs.
It is known that reduced coenzyme Q10 can be prepared by producing coenzyme Q10 in the conventional manner, for example by synthesis, fermentation, or extraction from natural products, and concentrating a reduced coenzyme Q10-containing eluate fraction resulting from chromatography (JP-A-10-109933). On that occasion, as described in the above-cited publication, the chromatographic concentration may be carried out after reduction of oxidized coenzyme Q10 contained in the reduced coenzyme Q10 as an impurity with a conventional reducing agent such as sodium borohydride or sodium dithionite (sodium hyposulfite), or reduced coenzyme Q10 may be prepared by reacting an existing highly pure grade of coenzyme Q10 with the reducing agent mentioned above. Additionally, a method is also known which comprises using zinc as a reducing agent (Journal of Lavelled Compounds, vol.6, 1970, 66-75). However, the above-mentioned methods of producing reduced coenzyme Q10 are not necessarily satisfiable. For example, a method comprising using chromatography is complicated for a commercial scale application. And the above reducing agent has problems such as generation of gas (hydrogen, sulfur dioxide, etc.), bad smell, issue of safety, treatment difficulty after use, and handling difficulty when it is applied on a commercial scale or when producing reduced coenzyme Q10 used for foods, functional nutritive foods, specific health foods, nutritional supplements, nutrients, animal drugs, drinks, feeds, cosmetics, medicines, remedies, preventive drugs, etc., thus is not necessarily preferable. A method for obtaining reduced coenzyme Q10 by the above reducing agent after obtaining a fraction of coenzyme Q10 by chromatography is not more unpreferable as a commercial production method. When the process and aftertreatment are complicated, deterioration in quality occurs as a result of the after-mentioned oxidation by a molecular oxygen.
Furthermore, in isolation of reduced coenzyme Q10 obtained by the above-mentioned method, it is not necessarily easy to isolate it in the state of high purity due to instability of reduced coenzyme Q10 for a molecular oxygen. In many cases, for example, the reduced coenzyme Q10 tends to occur as a low-purity crystalline, a semisolid or an oil containing such impurities as oxidized coenzyme Q10. As described above, it is very difficult to obtain a reduced coenzyme Q10 crystal of high quality even if a reaction mixture of reduced coenzyme Q10 containing completely or almost no oxidized coenzyme Q10 could be obtained by a reduction reaction.
Thus, it is a very important subject to stabilize reduced coenzyme Q10, namely to protect reduced coenzyme Q10 against oxidation. However, since reduced coenzyme Q10 has not commercially been available so far, there has hardly been any research on a method for stably retaining reduced coenzyme Q10 or the like method. Only WO 01/52822 A1 describes a composition containing a reducing agent and a production method thereof.
The above WO 01/52822 A1 discloses a method for producing reduced coenzyme Q10 which comprises reduction using various reducing agents such as vitamin C (i.e. ascorbic acid or related compounds such as ascorbic acid, ascorbyl palmitate and ascorbyl stearate) and vitamin E as more preferable reducing agents which may be used for foods, etc. The specification also discloses a composition comprising reduced coenzyme Q10, a reducing agent, and a surfactant, a vegetable oil or a mixture of these, and a composition for oral administration which is prepared in the form of a gelatin capsule or a tablet. Furthermore, the specification also discloses an in situ preparation method comprising using oxidized coenzyme Q10 and a reducing agent as a method for obtaining said compositions.
The above composition and the preparation method thereof are complicated and cumbersome. It is presumably because the above composition is expected to have plural roles (i.e. firstly, a composition to be a reaction field where oxidized coenzyme Q10 is reduced to reduced coenzyme Q10, and secondly, a composition which stably retains reduced coenzyme Q10). In other words, the above composition and the preparation method thereof makes it possible to reduce oxidized coenzyme Q10 to reduced coenzyme Q10 under a highly specific environment, as well as to stably retain the obtained reduced coenzyme Q10. However, the above method comprises reduction in the presence of components having a high boiling point or fat-soluble components, such as surfactants and vegetable oils, and it is very difficult to isolate reduced coenzyme Q10 after a reduction reaction. Therefore, applications of the above stabilization method and the composition are substantially limited to direct uses for foods, etc. The above method is an in situ preparation capable of retaining reduced coenzyme Q10 in pure state only in a reaction mixture.
The above WO 01/52822 A1 describes that compositions disclosed therein may contain, for example, as a solvent, an organic solvent such as a polyhydric alcohol, i.e. glycerine, 1,2-propanediol (propylene glycol) or the like, and ethanol in 0.25 to 50% by weight, preferably 1 to 25% by weight, more preferably 1.5 to 15-20% by weight if necessary. However, the above polyhydric alcohols and ethanol are not essential components. And, among Examples in said specification, Example 2 describes a composition without containing these solvents, Example 4 describes a composition containing 1.63% by weight of glycerine or propylene glycol, and Examples 1 and 3 respectively describe compositions each containing 4% by weight and 3.55% by weight of glycerine.
As a result of preliminary investigations on stabilization of reduced coenzyme Q10, the present inventors found that vitamin C has a stabilization effect whereas vitamin E does not have the effect, and a stabilization effect becomes very poor when vitamin C is used with polyhydric alcohols having 3 or more OH, such as glycerine, in combination.
In the above WO 01/52822 A1, there is no detail description on quality, stabilization effect, etc. of reduced coenzyme Q10 contained in the composition. And also there is no disclosure that a combination of vitamin C and a mono- and/or dihydric alcohol, especially a combination of vitamin C and a monohydric alcohol, exerts a significantly excellent stabilization effect. Furthermore, there is no description on a crystallization method, a composition, handling or preservation (including long-term stable preservation within an possible temperature range in ordinary conditions) which utilize the stabilization effect obtained by combinedly using vitamin C and a mono- and/or dihydric alcohol.
Thus, conventional methods were not necessarily satisfiable in producing reduced coenzyme Q10 by reducing oxidized coenzyme Q10, and in stably preserving it. Under such circumstances, it has been desired for developing a highly versatile stabilization method which overcomes the above-mentioned problems, and a preservation method, an isolation (crystallization) method and a composition using said stabilization method. Moreover, it has also been desired for developing a production method readily used for various applications by which reduced coenzyme Q10 of high quality may be obtained not only as a reaction mixture, but also preferably as a crystal.